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Viruses Feb 2021Lentiviral vectors (LVs) are potent tools for the delivery of genes of interest into mammalian cells and are now commonly utilised within the growing field of cell and... (Review)
Review
Lentiviral vectors (LVs) are potent tools for the delivery of genes of interest into mammalian cells and are now commonly utilised within the growing field of cell and gene therapy for the treatment of monogenic diseases and adoptive therapies such as chimeric antigen T-cell (CAR-T) therapy. This is a comprehensive review of the individual bioprocess operations employed in LV production. We highlight the role of envelope proteins in vector design as well as their impact on the bioprocessing of lentiviral vectors. An overview of the current state of these operations provides opportunities for bioprocess discovery and improvement with emphasis on the considerations for optimal and scalable processing of LV during development and clinical production. Upstream culture for LV generation is described with comparisons on the different transfection methods and various bioreactors for suspension and adherent producer cell cultivation. The purification of LV is examined, evaluating different sequences of downstream process operations for both small- and large-scale production requirements. For scalable operations, a key focus is the development in chromatographic purification in addition to an in-depth examination of the application of tangential flow filtration. A summary of vector quantification and characterisation assays is also presented. Finally, the assessment of the whole bioprocess for LV production is discussed to benefit from the broader understanding of potential interactions of the different process options. This review is aimed to assist in the achievement of high quality, high concentration lentiviral vectors from robust and scalable processes.
Topics: Animals; Bioreactors; Cell Culture Techniques; Cell Line; Genetic Vectors; Humans; Lentivirus; Transduction, Genetic; Viral Envelope Proteins; Virus Cultivation
PubMed: 33572347
DOI: 10.3390/v13020268 -
International Journal of Molecular... Apr 2023Gene therapy is a technique involving the modification of an individual's genes for treating a particular disease. The key to effective gene therapy is an efficient... (Review)
Review
Gene therapy is a technique involving the modification of an individual's genes for treating a particular disease. The key to effective gene therapy is an efficient carrier delivery system. Viral vectors that have been artificially modified to lose their pathogenicity are used widely as a delivery system, with the key advantages of their natural high transduction efficiency and stable expression. With decades of development, viral vector-based gene therapies have achieved promising clinical outcomes. Currently, the three key vector strategies are based on adeno-associated viruses, adenoviruses, and lentiviruses. However, certain challenges, such as immunotoxicity and "off-target", continue to exist. In the present review, the above three viral vectors are discussed along with their respective therapeutic applications. In addition, the major translational challenges encountered in viral vector-based gene therapies are summarized, and the possible strategies to address these challenges are also discussed.
Topics: Genetic Therapy; Genetic Vectors; Lentivirus; Adenoviridae; Gene Transfer Techniques; Dependovirus
PubMed: 37175441
DOI: 10.3390/ijms24097736 -
Neuroscience Letters Aug 2019Microglia have emerged as a critical component of neurodegenerative diseases. Genetic manipulation of microglia can elucidate their functional impact in disease. In... (Review)
Review
Microglia have emerged as a critical component of neurodegenerative diseases. Genetic manipulation of microglia can elucidate their functional impact in disease. In neuroscience, recombinant viruses such as lentiviruses and adeno-associated viruses (AAVs) have been successfully used to target various cell types in the brain, although effective transduction of microglia is rare. In this review, we provide a short background of lentiviruses and AAVs, and strategies for designing recombinant viral vectors. Then, we will summarize recent literature on successful microglial transductions in vitro and in vivo, and discuss the current challenges. Finally, we provide guidelines for reporting the efficiency and specificity of viral targeting in microglia, which will enable the microglial research community to assess and improve methodologies for future studies.
Topics: Animals; Dependovirus; Gene Targeting; Genetic Vectors; HIV-1; Humans; Lentivirus; Microglia; Transduction, Genetic
PubMed: 31158432
DOI: 10.1016/j.neulet.2019.134310 -
Nature Protocols Apr 2022Human intestinal tissue-derived enteroids (HIEs; also called organoids) are a powerful ex vivo model for gastrointestinal research. Genetic modification of these... (Review)
Review
Human intestinal tissue-derived enteroids (HIEs; also called organoids) are a powerful ex vivo model for gastrointestinal research. Genetic modification of these nontransformed cultures allows new insights into gene function and biological processes involved in intestinal diseases as well as gastrointestinal and donor segment-specific function. Here we provide a detailed technical pipeline and protocol for using the CRISPR-Cas9 genome editing system to knock out a gene of interest specifically in HIEs by lentiviral transduction and single-cell cloning. This protocol differs from a previously published alternative using electroporation of human colonoids to deliver piggyback transposons or CRISPR-Cas9 constructs, as this protocol uses a modified, fused LentiCRISPRv2-small-guiding RNA to express Cas9 and small-guiding RNA in a lentivirus. The protocol also includes the steps of gene delivery and subsequent single-cell cloning of the knockout cells as well as verification of clones and sequence identification of the mutation sites to establish knockout clones. An overview flowchart, step-by-step guidelines and troubleshooting suggestions are provided to aid the researcher in obtaining the genetic knockout HIE line within 2-3 months. In this protocol, we further describe how to use HIEs as an ex vivo model to assess host restriction factors for viral replication (using human norovirus replication as an example) by knocking out host attachment factors or innate immunity genes. Other applications are discussed to broaden the utility of this system, for example, to generate knockin or conditional knockout HIE lines to investigate the function of essential genes in many biological processes including other types of organoids.
Topics: CRISPR-Cas Systems; Clone Cells; Cloning, Molecular; Gene Editing; Gene Knockout Techniques; Humans; Lentivirus; RNA, Guide, CRISPR-Cas Systems
PubMed: 35197604
DOI: 10.1038/s41596-021-00669-0 -
Nature Protocols Aug 2020Massively parallel reporter assays (MPRAs) can simultaneously measure the function of thousands of candidate regulatory sequences (CRSs) in a quantitative manner. In...
Massively parallel reporter assays (MPRAs) can simultaneously measure the function of thousands of candidate regulatory sequences (CRSs) in a quantitative manner. In this method, CRSs are cloned upstream of a minimal promoter and reporter gene, alongside a unique barcode, and introduced into cells. If the CRS is a functional regulatory element, it will lead to the transcription of the barcode sequence, which is measured via RNA sequencing and normalized for cellular integration via DNA sequencing of the barcode. This technology has been used to test thousands of sequences and their variants for regulatory activity, to decipher the regulatory code and its evolution, and to develop genetic switches. Lentivirus-based MPRA (lentiMPRA) produces 'in-genome' readouts and enables the use of this technique in hard-to-transfect cells. Here, we provide a detailed protocol for lentiMPRA, along with a user-friendly Nextflow-based computational pipeline-MPRAflow-for quantifying CRS activity from different MPRA designs. The lentiMPRA protocol takes ~2 months, which includes sequencing turnaround time and data processing with MPRAflow.
Topics: Base Sequence; Lentivirus; Regulatory Sequences, Nucleic Acid; Sequence Analysis, DNA; Workflow
PubMed: 32641802
DOI: 10.1038/s41596-020-0333-5 -
Nature Methods Apr 2018Several groups recently coupled CRISPR perturbations and single-cell RNA-seq for pooled genetic screens. We demonstrate that vector designs of these studies are...
Several groups recently coupled CRISPR perturbations and single-cell RNA-seq for pooled genetic screens. We demonstrate that vector designs of these studies are susceptible to ∼50% swapping of guide RNA-barcode associations because of lentiviral template switching. We optimized a published alternative, CROP-seq, in which the guide RNA also serves as the barcode, and here confirm that this strategy performs robustly and doubled the rate at which guides are assigned to cells to 94%.
Topics: CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Lentivirus; RNA, Guide, CRISPR-Cas Systems; Sequence Analysis, RNA; Single-Cell Analysis
PubMed: 29457792
DOI: 10.1038/nmeth.4604 -
RNA (New York, N.Y.) Apr 2003Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila...
Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila melanogastor, and Caenorhabditis elegans, but similar techniques have proven difficult to apply to mammalian systems. Although manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics. Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP) using Moloney leukemia virus-based and lentivirus-based retroviral vectors. Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts. This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells.
Topics: Dendritic Cells; Genes, Reporter; HeLa Cells; Humans; In Vitro Techniques; Lentivirus; RNA Interference
PubMed: 12649500
DOI: 10.1261/rna.2192803 -
Nature Methods Jun 2011Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss-of-function and gain-of-function experiments....
Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss-of-function and gain-of-function experiments. We describe the production of a sequence-confirmed, clonal collection of over 16,100 human open-reading frames (ORFs) encoded in a versatile Gateway vector system. Using this ORFeome resource, we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.
Topics: Cloning, Molecular; Genetic Vectors; Genomic Library; Humans; Lentivirus; Open Reading Frames
PubMed: 21706014
DOI: 10.1038/nmeth.1638 -
Blood Advances Nov 2020Genetic modification of T lymphocytes is a key issue in research and therapy. Conventional lentiviral vectors (LVs) are neither selective for T cells nor do they modify...
Genetic modification of T lymphocytes is a key issue in research and therapy. Conventional lentiviral vectors (LVs) are neither selective for T cells nor do they modify resting or minimally stimulated cells, which is crucial for applications, such as efficient in vivo modification of T lymphocytes. Here, we introduce novel CD3-targeted LVs (CD3-LVs) capable of genetically modifying human T lymphocytes without prior activation. For CD3 attachment, agonistic CD3-specific single-chain variable fragments were chosen. Activation, proliferation, and expansion mediated by CD3-LVs were less rapid compared with conventional antibody-mediated activation owing to lack of T-cell receptor costimulation. CD3-LVs delivered genes not only selectively into T cells but also under nonactivating conditions, clearly outperforming the benchmark vector vesicular stomatitis-LV glycoproteins under these conditions. Remarkably, CD3-LVs were properly active in gene delivery even when added to whole human blood in absence of any further stimuli. Upon administration of CD3-LV into NSG mice transplanted with human peripheral blood mononuclear cells, efficient and exclusive transduction of CD3+ T cells in all analyzed organs was achieved. Finally, the most promising CD3-LV successfully delivered a CD19-specific chimeric antigen receptor (CAR) into T lymphocytes in vivo in humanized NSG mice. Generation of CAR T cells was accompanied by elimination of human CD19+ cells from blood. Taken together, the data strongly support implementation of T-cell-activating properties within T-cell-targeted vector particles. These particles may be ideally suited for T-cell-specific in vivo gene delivery.
Topics: Animals; Genetic Vectors; Lentivirus; Leukocytes, Mononuclear; Mice; T-Lymphocytes; Transduction, Genetic
PubMed: 33216892
DOI: 10.1182/bloodadvances.2020002229 -
Viruses Jul 2022Lentiviral vectors (LV) have been developed upon knowledge accumulated in the virology field, in particular intensive research on HIV biology since its discovery in 1983...
Lentiviral vectors (LV) have been developed upon knowledge accumulated in the virology field, in particular intensive research on HIV biology since its discovery in 1983 [...].
Topics: Genetic Vectors; Lentivirus
PubMed: 35891475
DOI: 10.3390/v14071492